This proposal targets the molecular basis of iron overload disorders and iron-restricted anemias which are among the most common hematological diseases worldwide. The molecular mechanisms of iron homeostasis or its disorders were not understood. Recent studies, many from our laboratory, have identified the iron- regulatory hormone hepcidin and its receptor/iron efflux channel ferroportin as the key molecules responsible for the regulation of extracellular iron concentration and systemic iron homeostasis. The 25 amino acid peptide hepcidin is secreted by hepatocytes and binds to ferroportin in cells that export iron to plasma: duodenal enterocytes that absorb dietary iron, macrophages that recycle senescent red cells, and hepatocytes that store iron. Upon binding hepcidin, ferroportin is internalized and degraded, and cellular iron export into plasma decreases. Hepcidin deficiency results in excessive absorption and release of iron into extracellular fluid, causing iron overload in hereditary hemochromatoses, and causing or contributing to iron overload in iron-loading anemias. Hepcidin excess causes the anemia of inflammation, and iron-refractory iron-deficiency anemia. Hepcidin agonists and antagonists could be used to treat these disorders. We propose to analyze the structural basis of the hepcidin-ferroportin interaction, by extensively mutating the interacting molecular structures. In a synergistic parallel effort, we will develop potent hepcidin agonists by optimization of existing bioactive 6-9 amino acid peptides, and by modifying a successful high throughput screen for antagonists to detect small molecules with agonist activity. Specifically we will: 1) Characterize the key elements of hepcidin structure required for activity 2) Identify the ferroportin structure required for the interaction with hepcidin 3) Examine the agonist and antagonist activity of minihepcidins 4) Undertake a high throughput screen for small molecules with hepcidin agonist activity Successful completion of these studies will not only increase our understanding of this newly recognized homeostatic mechanism but also lay the foundation for translating these advances into tangible benefits for patients with iron disorders. PUBLIC HEALTH RELEVANCE: The proposed project will help understand and treat disorders of iron metabolism including hemochromatosis and thalassemias.